PROJECT SUMMARY Autophagy is an essential homeostatic process. Under stressful conditions, such as nutrient deprivation, autophagy is markedly up-regulated to provide a source of raw materials to ensure survival and to dispose of damaged organelles. Autophagosomes are generated by elongation and closure of phagophores, and autophagosomes fuse with lysosomes to effect content degradation and recycling. Phosphoinositide lipids have been implicated in every stage of the autophagic pathway. However, to date, emphasis has been placed on the function of PI3P in phagophore elongation and closure. We found that PI4P is required for autophagosome fusion with lysosomes (A:L fusion), a culminating event at the end of the autophagy road. Furthermore, PI4K2A (PI4KII?) is uniquely responsible for generating the PI4P on autophagosomes and it acts downstream of GABARAPs, Atg8 family autophagy proteins that are much less well understood than the LC3 Atg8 family. PI4K2A binds GABARAPs and associates with autophagosomes in a GABARAP- and palmitoylation-dependent manner. It is activated by starvation and translocates from the perinuclear Golgi region to autophagosomes. PI4K2A or GABARAP depletion blocks autophagy flux and A:L fusion, resulting in the formation of abnormally large autophagosomes. This block can be rescued by overexpressing PI4K2A or increasing intracellular PI4P by ?PI4P shuttling.? We hypothesize that GABARAP promotes PI4K2A membrane trafficking to autophagosomes to generate PI4P in the GABARAP/PI4K2A interactome to activate the A:L fusion machinery. This hypothesis will be tested as follows: Aim I. Determine how GABARAPs target PI4K2A to autophagosomes. We will analyze PI4P dynamics and the temporal sequence of PI4K2A recruitment to autophagosomes. We will determine if GABARAP acts as a trafficking modulator to promote PI4K2A recruitment, and identify/disrupt the binding motifs to establish functionality of the interaction. Aim 2. Determine how PI4K2A and PI4P promote A:L fusion. We will focus on the role of PI4K2A in the recruitment and activation of Rab7, the master regulator of the A:L priming, tethering and fusion. We will test the hypothesis that Rab7 is targeted to autophagosomes by direct binding to PI4K2A or PI4P and/or by PI4P-mediated recruitment of the multivalent scaffolding protein, PLEKHM1. Mechanisms whereby PI4K2A/PI4P control the activity state of Rab7 will be examined. Aim 3. Determine how PI4K2A is regulated during autophagy. The effects of autophagy-dependent changes in PI4K2A phosphorylation and palmitoylation on targeting and kinase activity will be examined. A combination of biochemical, cell biological, and state-of-the-art fluorescence-based nanoimaging approaches will be employed to address these questions. This multi-PI grant will provide a comprehensive picture of the function and regulation of PI4K2A in its hitherto unexplored role in autophagy. In doing so, it will establish a novel paradigm for the regulation of autophagy and will address pressing problems in many human diseases associated with defective autophagy.